1. Field of the Invention
This invention pertains generally to microfluidic devices and diagnostic schemes, and more particularly to a microfluidic imaging platform for radioactivity-based biological activity assays on protein or small molecule substrates.
2. Description of Related Art
The observation of abnormal biological activity can be an important diagnostic tool for determining the presence of human diseases as well as a focus for treatment and the study of diseases. Pharmaceuticals directed to promoting or inhibiting certain physiological functions have provided relief of symptoms as well as the successful treatment of many diseases. For example, the 518 known kinases in humans constitute nearly two percent of the human genome and represent one of the largest classes of drug targets pursued by pharmaceutical companies. With as many as 50% of intracellular proteins regulated by phosphorylation-mediated signal transduction, kinases play a major role in numerous aspects of cell biology, including proliferation, differentiation, secretion and apoptosis. Kinase dysfunction has been implicated in a wide variety of diseases including many cancers.
Kinase-related signaling measurements that are taken directly from patient samples often involve detecting the consequences of activity, for example the resulting downstream phosphorylation patterns. Direct measurement of kinase activity from patient samples can greatly complement these phosphoprofiling techniques.
The broad interest in targeting kinases for drug discovery has lead to the development of numerous kinase assay technologies. Among them, radiometric assays represent the earliest kinase assay technology and are generally considered the “gold standard” for determining basic enzyme properties. These assays utilize ATP radiolabeled on the γ-phosphate (generally 32P or 33P). In a kinase reaction, the radioisotope is transferred from ATP to the acceptor molecule, and the rate of product formation can be quantified by measuring the extent of isotope incorporation. Radiometric kinase assays are useful for determining basic enzyme properties, in part, because radiolabeling of reaction substrates does not alter their intrinsic biochemical and physical properties.
Although they are adopted as the primary technology for kinase profiling services, all radiometric assays suffer from the same drawback in that they are difficult to miniaturize. The requirement of a sufficient number of input cells makes it difficult to study kinase activity on small volume samples. For example, BCR-ABL is a constitutively activated, oncogenic tyrosine kinase that causes both chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL inhibitors, such as imatinib (Gleevec) are widely used in clinical applications but patients often acquire drug resistance at some point during the treatment. One type of drug resistance is caused by the natural or developed insensitivity of leukemic stem cells to kinase inhibitors. The urgent need for studying kinase activity in patient cancer samples and in cancer stem cells cannot be fully met by conventional radiometric assays due to the limited amount of sample that is available. Thus far, little has been done to miniaturize the in vitro kinase radioassay beyond the 96 well format to make it compatible with limited sample size.
In addition, many fluorescence/luminescence-based assays have emerged to avoid exposure to radioactivity. However, fluorescence assays have their own set of drawbacks including the requirement of a fair amount of cell input, being less quantitative than radiometric kinase assays, and often requiring engineered substrates to be created for each particular kinase. Therefore, the conventional kinase assay is inadequate for evaluation and imaging of small sample sizes.
Accordingly, there is a need for an assay apparatus and method that can provide a reliable, highly sensitive analysis and assay a variety of different kinase or other biological activity without the need for a large sample size. The present invention satisfies this need as well as others and is an improvement over the art.